Chromatin Immunoprecipitation Data Research Articles

Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity.

Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data, revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes.

Taking the Wheel - de novo DNA Methylation as a Driving Force of Plant Embryonic Development.

During plant embryogenesis, regardless of whether it begins with a fertilized egg cell (zygotic embryogenesis) or an induced somatic cell (somatic embryogenesis), significant epigenetic reprogramming occurs with the purpose of parental or vegetative transcript silencing and establishment of a next-generation epigenetic patterning. To ensure genome stability of a developing embryo, large-scale transposon silencing occurs by an RNA-directed DNA methylation (RdDM) pathway, which introduces methylation patterns de novo and as such potentially serves as a global mechanism of transcription control during developmental transitions. RdDM is controlled by a two-armed mechanism based around the activity of two RNA polymerases. While PolIV produces siRNAs accompanied by protein complexes comprising the methylation machinery, PolV produces lncRNA which guides the methylation machinery toward specific genomic locations. Recently, RdDM has been proposed as a dominant methylation mechanism during gamete formation and early embryo development in Arabidopsis thaliana, overshadowing all other methylation mechanisms. Here, we bring an overview of current knowledge about different roles of DNA methylation with emphasis on RdDM during plant zygotic and somatic embryogenesis. Based on published chromatin immunoprecipitation data on PolV binding sites within the A. thaliana genome, we uncover groups of auxin metabolism, reproductive development and embryogenesis-related genes, and discuss possible roles of RdDM at the onset of early embryonic development via targeted methylation at sites involved in different embryogenesis-related developmental mechanisms.

Myogenic differentiation 1 and transcription factor 12 activate the gene expression of mouse taste receptor type 1 member 1

ObjectivesMyogenic differentiation 1 (Myod1) is involved in the expression of taste receptor type 1 member 1 (Tas1r1) during myogenic differentiation. Further, the target genes of Myod1 participate in transcriptional control, muscle development, and synaptic function. We examined, for the first time, the function of Myod1 in the transcriptional regulation of Tas1r1. MethodsENCODE chromatin immunoprecipitation and sequencing (ChIP-seq) data of myogenically differentiated C2C12 cells were analyzed to identify the Myod1 and transcription factor 12 (Tcf12) binding sites in the Tas1r1 promoter region. Luciferase reporter assays, DNA affinity precipitation assays, and co-immunoprecipitation assays were also performed to identify the functions of Myod1, Tcf12, and Krüppel-like factor 5 (Klf5). ResultsBased on ENCODE ChIP-seq, Myod1 bound to the Tas1r1 promoter region containing E-boxes 1–3. Luciferase reporter assays revealed that site-directed E-box1 mutations significantly reduced promoter activation induced by Myod1 overexpression. According to the DNA affinity precipitation assay and co-immunoprecipitation assay, Myod1 formed a heterodimer with Tcf12 and bound to E-box1. Further, Klf5 bound to the GT box near E-box1, activating Tas1r1 expression. ConclusionsDuring myogenic differentiation, the Myod1/Tcf12 heterodimer, in collaboration with Klf5, binds to E-box1 and activates Tas1r1 expression.

The conserved Tpk1 regulates non-homologous end joining double-strand break repair by phosphorylation of Nej1, a homolog of the human XLF.

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, encompassing three catalytic (Tpk1–3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.

Characterization of expansin genes and their transcriptional regulation by histone modifications in strawberry.

The possible candidate expansin genes, which may be important for strawberry fruit softening, have been identified in the diploid woodland strawberry Fragaria vesca and the octoploidcultivated strawberry Fragaria × ananassa and their transcriptional regulation by histone modifications has been studied. Softening process greatly affects fruit texture and shelf life. Expansins (EXPs) are a group of structural proteins participating in cell wall loosening, which break the hydrogen bonding between cellulose microfibrils and hemicelluloses. However, our knowledge on how EXP genes are regulated in fruit ripening, especially in non-climacteric fleshy fruits, is limited. Here, we have identified the EXP genes in both the octoploidcultivated strawberry (Fragaria × ananassa) and one of its diploid progenitor species, woodland strawberry (Fragaria vesca). We found that EXP proteins in F. × ananassa were structurally more divergent than the ones in F. vesca. Transcriptome data suggested that FaEXP88, FaEXP114, FveEXP11 and FveEXP33 were the four candidate EXP genes more likely involved in fruit softening, whose transcript levels dramatically increased when firmness decreased during fruit maturation. Phylogenetic analyses showed that those candidate genes were closely clustered, indicating the presence of homoeolog expression dominance in the EXP gene family in strawberry. Moreover, we have performed chromatin immunoprecipitation (ChIP) experiments to investigate the distribution of histone modifications along the promoters and genic regions of the EXP genes in F. vesca. ChIP data revealed that the transcript levels of EXP genes were highly correlated with the enrichment of H3K9/K14 acetylation and H3K27 tri-methylation. Collectively, this study identifies the key EXP genes involved in strawberry fruit softening and reveals a regulatory role of histone modifications in their transcriptional regulation, which would facilitate functional studies of the EXP genes in the ripening of non-climacteric fruits.

Abstract 2119: Genome-wide epigenetic alterations in T-cell prolymphocytic leukemia

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease showing a rapid clinical course, with a median survival of <1 year. Whole-genome and whole-exome sequencing have identified key genetic lesions in T-PLL, including structural alterations such as inversions, translocations, and large copy number variants, as well as recurrent somatic mutations of genes encoding chromatin regulators and of those in the JAK-STAT signaling pathway. Epigenetic alteration is implicated in the susceptibility and progression of both cancer and non-cancerous diseases. However, genome-wide epigenetic data has not been generated for T-PLL, which prohibits the mechanistic studies of transcriptional misregulation in T-PLL. This study used micrococcal nuclease digestion and sequencing (MNase-seq) to profile the chromatin accessible regions, i.e., gene regulatory regions such as promoters, enhancers and insulators, in both T-PLL patients and healthy individuals. Our analysis revealed the prevalence of lost open chromatin regions in T-PLL. We also generated histone H3 lysine 4 monomethylation (H3K4me1) and lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation and sequencing (ChIP-seq) data from the same samples. We identified 5,462 and 2,833 active enhancers (>=2.5 kb away from transcription start sites) that are lost and gained in T-PLL, respectively, compared to normal. The differential H3K27ac peaks are preferentially associated with the nearest genes differentially expressed between T-PLL and normal (chi-square test p <2.2e-16), indicating a role of epigenetic alteration in transcriptional misregulation. In addition, we identified alterations of active enhancers that target oncogenes with key roles in T-PLL, such as TCL1Aand MYC. Together, our analysis has provided insights into the epigenetic mechanisms that drive oncogenic activation in T-PLL. Citation Format: Shulan Tian, Henan Zhang, Eric Klee, Huihuang Yan, Wei Ding. Genome-wide epigenetic alterations in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2119.

Two Arabidopsis Homologs of Human Lysine-Specific Demethylase Function in Epigenetic Regulation of Plant Defense Responses.

Epigenetic marks such as covalent histone modification and DNA methylation are crucial for mitotically and meiotically inherited cellular memory-based plant immunity. However, the roles of individual players in the epigenetic regulation of plant immunity are not fully understood. Here we reveal the functions of two Arabidopsis thaliana homologs of human lysine-specific demethylase1-like1, LDL1 and LDL2, in the maintenance of methyl groups at lysine 4 of histone H3 and in plant immunity to Pseudomonas syringae infection. The growth of virulent P. syringae strains was reduced in ldl1 and ldl2 single mutants compared to wild-type plants. Local and systemic disease resistance responses, which coincided with the rapid, robust transcription of defense-related genes, were more stably expressed in ldl1 ldl2 double mutants than in the single mutants. At the nucleosome level, mono-methylated histone H3K4 accumulated in ldl1 ldl2 plants genome-wide and in the mainly promoter regions of the defense-related genes examined in this study. Furthermore, in silico comparative analysis of RNA-sequencing and chromatin immunoprecipitation data suggested that several WRKY transcription factors, e.g., WRKY22/40/70, might be partly responsible for the enhanced immunity of ldl1 ldl2. These findings suggest that LDL1 and LDL2 control the transcriptional sensitivity of a group of defense-related genes to establish a primed defense response in Arabidopsis.

174-OR: ADA Presidents’ Select Abstract: Epigenetic Regulation of Functional Beta-Cell Mass by Cohesin Smc3

Both type 1 and type 2 diabetes are caused by insulin insufficiency due to inadequate functional beta-cell mass. The demise of beta-cells in diabetes is preceded by progressive impairment of beta-cell function. Several studies have shown that the regulation of beta-cell function and survival is tightly linked. However, the precise molecular mechanisms that link beta-cell function and survival remain far from clear. In this study, we report that the cohesin Smc3 is a novel epigenetic regulator of functional beta-cell mass. Smc3 is an ATPase that regulates the 3D chromatin architecture and chromatid cohesion, and plays an important role in cellular self-renewal, differentiation, and survival. Smc3 exists in a tripartite ring complex with proteins Smc1 and Rad21, and the association of this complex to the chromatin is regulated by Stag1. Our chromatin immunoprecipitation data suggest that the cohesin complex regulates beta-cell maturation and survival programs. Using transcriptomic profiling combined with histological and physiological analyses, we show that that the beta-cell specific Smc3 knockout mice (Smc3-BKO) become diabetic by 3 months of age, due to a progressive loss of beta-cell maturity and viability. Islets from the Smc3-BKO mice display impaired glucose stimulated insulin secretion (GSIS) around 1-2 months of age, with gene expression signatures that resemble functionally immature neonatal beta-cells. This is followed by progressive decline of beta-cell mass. At a molecular level, disruption of Smc3 leads to reduced protein levels of the other two subunits of the cohesin ring complex, Smc1 and Rad21. On the other hand, the mRNA levels of Stag1 are increased in the Smc3-BKO islets. Finally, we show that the cohesin regulatory program is disrupted in islets from humans with type 2 diabetes. Taken together, these studies provide a novel insight into the integrative epigenetic regulation of functional beta-cell mass. Disclosure J. K. Wang: None. A. Pandey: None. A. P. Ham: None. N. Parveen: None. S. Dhawan: None.

Single loss of a Trp53 allele triggers an increased oxidative, DNA damage and cytokine inflammatory responses through deregulation of I\u03baB\u03b1 expression

Dose of Trp53, the main keeper of genome stability, influences tumorigenesis; however, the causes underlying and driving tumorigenesis over time by the loss of a single p53 allele are still poorly characterized. Here, we found that single p53 allele loss specifically impacted the oxidative, DNA damage and inflammatory status of hematopoietic lineages. In particular, single Trp53 allele loss in mice triggered oxidative stress in peripheral blood granulocytes and spleenocytes, whereas lack of two Trp53 alleles produced enhanced oxidative stress in thymus cells, resulting in a higher incidence of lymphomas in the Trp53 knockout (KO) mice compared with hemizygous (HEM). In addition, single or complete loss of Trp53 alleles, as well as p53 downregulation, led to a differential increase in basal, LPS- and UVB-induced expression of a plethora of pro-inflammatory cytokine, such as interleukin-12 (Il-12a), TNFα (Tnfa) and interleukin (Il-23a) in bone marrow-derived macrophage cells (BMDMs) compared to WT cells. Interestingly, p53-dependent increased inflammatory gene expression correlated with deregulated expression of the NF-κB pathway inhibitor IκBα. Chromatin immunoprecipitation data revealed decreased p65 binding to Nfkbia in the absence of p53 and p53 binding to Nfkbia promoter, uncovering a novel crosstalk mechanism between p53 and NF-κB transcription factors. Overall, our data suggest that single Trp53 allele loss can drive a sustained inflammatory, DNA damage and oxidative stress response that, over time, facilitate and support carcinogenesis.

WHSC1/NSD2 regulates immune infiltration in prostate cancer

BackgroundImmunotherapy in prostate cancer (PCa) lags behind the progresses obtained in other cancer types partially because of its limited immune infiltration. Tumor-resident immune cells have been detected in the prostate,...

TBIO-16. NOTCH1 PATHWAY AS TARGET FOR DRUG INTERVENTION FOR HISTONE 3 G34R MUTATED pHGG

There have been no significant improvements in the treatments for childhood High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), which continue to have a very poor prognosis. These cancers harbor mutations affecting histone 3 (H3) proteins; 80% of DIPGs with histone H3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. We have generated and validated a histone 3.3 G34R mutant-specific antibody and investigated the downstream effects of H3.3 G34R mutations in pHGG. In order to identify the genes that may be deregulated by G34R mutant histone expression, we have performed chromatin immunoprecipitation (ChIP) assays with our H3.3 G34R and wild type H3 antibodies, using pHGG H3 G34R mutant and wild-type cell lines. Initial analyses of ChIP data have implicated deregulation of cell-signaling pathways including Notch1, Hedgehog, PPAR-1, PLC-beta and Androgen, in H3 G34R mutated pHGG. We are currently determining the effects of altered expression of Notch1 pathway components on tumorigenesis of H3 G34R mutated pHGG, through gene and protein expression and inhibition assays. Specifically we find that the Notch1 pathway component HES1 shows increased expression in G34R mutant cells compared to controls, directing our evaluation of the utility of gamma-secretase inhibitors as potential therapeutics. These analyses may underpin development of novel treatment strategies for H3 mutated pHGG.

Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y

A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

A machine learning framework for the prediction of chromatin folding in Drosophila using epigenetic features.

Technological advances have lead to the creation of large epigenetic datasets, including information about DNA binding proteins and DNA spatial structure. Hi-C experiments have revealed that chromosomes are subdivided into sets of self-interacting domains called Topologically Associating Domains (TADs). TADs are involved in the regulation of gene expression activity, but the mechanisms of their formation are not yet fully understood. Here, we focus on machine learning methods to characterize DNA folding patterns in Drosophila based on chromatin marks across three cell lines. We present linear regression models with four types of regularization, gradient boosting, and recurrent neural networks (RNN) as tools to study chromatin folding characteristics associated with TADs given epigenetic chromatin immunoprecipitation data. The bidirectional long short-term memory RNN architecture produced the best prediction scores and identified biologically relevant features. Distribution of protein Chriz (Chromator) and histone modification H3K4me3 were selected as the most informative features for the prediction of TADs characteristics. This approach may be adapted to any similar biological dataset of chromatin features across various cell lines and species. The code for the implemented pipeline, Hi-ChiP-ML, is publicly available: https://github.com/MichalRozenwald/Hi-ChIP-ML

Abstract PO-31: HIV-1 transactivator of transcription deregulates key Burkitt lymphoma associated oncogenes at both the transcriptional and post-transcriptional level

Abstract Burkitt lymphoma (BL) is one of the most common HIV-associated lymphomas. This cancer represents one of the most frequent causes of mortality among HIV+ people in Southern Africa, which has the highest incidence of HIV/AIDS worldwide. As is the case for Kaposi sarcoma, recent reports associate an oncogenic function with HIV in lymphoma development. This study explores the oncogenic potential of HIV-1 protein Transactivator of transcription (Tat) in BL via its ability to manipulate the expression of c-MYC and AID, two key drivers of disease in BL. The ability of HIV-1 Tat to influence the activity of the full-length (FL) (2861 bp) c-MYC promoter was assessed using Dual Luciferase Reporter (DLR) assays. Using sequential deletions, the minimal promoter region mediating the effect was identified. Site-directed mutagenesis (SDM) was used to identify promoter elements mediating the response. Co-immunoprecipitation assays (Co-IP) were used to assess interactions between Tat and the AP-1 factor JunB. Chromatin Immunoprecipitation (ChIP) assays were done to confirm that JunB bound the promoter in vivo. To assess the ability of Tat to mediate the expression of miRNA-181b-5p, a known repressor of AICDA, Ramos cells were transfected with Tat followed by qRT-PCR. Corresponding changes in AID protein levels were determined using Western blot. To assess whether miRNA-181b-5p targets AICDA in B cells, the FL AICDA 3'UTR was cloned into the pGL3-Promoter vector and DLR assays were performed in the presence of miRNA-181b-5p mimic. Our data reveal that Tat enhances c-MYC promoter activity in a dose-dependent manner, with a maximum activity of 2.6-fold recorded when 500ng of pcDNA-Tat was used on 400ng of pGL3-c-MYC plasmid harboring the FL promoter. A loss of 35% activity was observed [1.7 (±0.05) fold] upon promoter deletion and two AP-1 sites (positions -1128 bp and -1376 bp) within the deleted region were found to mediate the Tat-dependent increase. A loss in activity of 20.6% was recorded when AP-1 site 1 was mutated, and 25% when AP-1 site 2 was mutated. A double mutant decreased the activity by 29%. Co-IP revealed a strong interaction between Tat and AP-1 factor JunB, and ChIP data showed that JunB was strongly recruited to those two AP-1 sites in the presence of Tat. Our result also reveals that the expression of the AICDA specific miRNA181b-5p is significantly reduced in the presence of Tat, which correlates with an increase in AID protein levels. We also confirm that the AICDA FL 3'UTR, which contains putative miRNA-181b-5p binding sites, is partially repressed by a miRNA-181b-5p mimic. Our study reveals that Tat potentiates oncogenesis by enhancing the expression of c-MYC and AID, two key lymphoma drivers. We show that Tat can enhance c-MYC expression by enhancing the recruitment of cellular TFs to the promoter. We also show that the same HIV protein can enhance the expression of AID by modulating the expression of miRNA-181b-5p. Citation Format: Leonardo Alves de Souza Rios, Nontlantla Mdletshe, Shaheen Mowla. HIV-1 transactivator of transcription deregulates key Burkitt lymphoma associated oncogenes at both the transcriptional and post-transcriptional level [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-31.

LINC00173.v1 promotes angiogenesis and progression of lung squamous cell carcinoma by sponging miR-511-5p to regulate VEGFA expression

BackgroundAnti-angiogenic therapy represents a promising strategy for non-small-cell lung cancer (NSCLC) but its application in lung squamous cell carcinoma (SQC) is limited due to the high-risk adverse effects. Accumulating evidence indicates that long noncoding RNAs (lncRNAs) mediate in tumor progression by participating in the regulation of VEGF in NSCLC, which might guide the development of new antiangiogenic strategies.MethodsDifferential lncRNA expression in SQC was analyzed in AE-meta and TCGA datasets, and further confirmed in lung cancer tissues and adjacent normal tissues with RT-qPCR and in-situ hybridization. Statistical analysis was performed to evaluate the clinical correlation between LINC00173.v1 expression and survival characteristics. A tube formation assay, chick embryo chorioallantoic membrane assay and animal experiments were conducted to detect the effect of LINC00173.v1 on the proliferation and migration of vascular endothelial cells and tumorigenesis of SQC in vivo. Bioinformatics analysis, RNA immunoprecipitation and luciferase reporter assays were performed to elucidate the downstream target of LINC00173.v1. The therapeutic efficacy of antisense oligonucleotide (ASO) against LINC00173.v1 was further investigated in vivo. Chromatin immunoprecipitation and high throughput data processing and visualization were performed to identify the cause of LINC00173.v1 overexpression in SQC.ResultsLINC00173.v1 was specifically upregulated in SQC tissues, which predicted poorer overall and progression-free survival in SQC patients. Overexpression of LINC00173.v1 promoted, while silencing LINC00173.v1 inhibited the proliferation and migration of vascular endothelial cells and the tumorigenesis of SQC cells in vitro and in vivo. Our results further revealed that LINC00173.v1 promoted the proliferation and migration of vascular endothelial cells and the tumorigenesis of SQC cells by upregulating VEGFA expression by sponging miR-511-5p. Importantly, inhibition of LINC00173.v1 via the ASO strategy reduced the tumor growth of SQC cells, and enhanced the therapeutic sensitivity of SQC cells to cisplatin in vivo. Moreover, our results showed that squamous cell carcinoma-specific factor ΔNp63α contributed to LINC00173.v1 overexpression in SQC.ConclusionOur findings clarify the underlying mechanism by which LINC00173.v1 promotes the proliferation and migration of vascular endothelial cells and the tumorigenesis of SQC, demonstrating that LINC00173.v1-targeted drug in combination with cisplatin may serve as a rational regimen against SQC.

The proteasome activator PA200 regulates expression of genes involved in cell survival upon selective mitochondrial inhibition in neuroblastoma cells.

The conserved Blm10/PA200 activators bind to the proteasome core and facilitate peptide and protein turnover. Blm10/PA200 proteins enhance proteasome peptidase activity and accelerate the degradation of unstructured proteasome substrates. Our knowledge about the exact role of PA200 in diseased cells, however, is still limited. Here, we show that stable knockdown of PA200 leads to a significantly elevated number of cells in S phase after treatment with the ATP synthase inhibitor, oligomycin. However, following exposure to the complex I inhibitor rotenone, more PA200‐depleted cells were in sub‐G1 and G2/M phases indicative of apoptosis. Chromatin immunoprecipitation (ChIP) and ChIP‐seq data analysis of collected reads indicate PA200‐enriched regions in the genome of SH‐SY5Y. We found that PA200 protein peaks were in the vicinity of transcription start sites. Gene ontology annotation revealed that genes whose promoters were enriched upon anti‐PA200 ChIP contribute to the regulation of crucial intracellular processes, including proliferation, protein modifications and metabolism. Selective mitochondrial inhibitors induced PA200 redistribution in the genome, leading to protein withdrawal from some gene promoters and binding to others. Collectively, the results support a model in which PA200 potentially regulates cellular homeostasis at the transcriptional level, in addition to its described role as an alternative activator of the proteasome.

An LRR-only protein regulates abscisic acid-mediated abiotic stress responses during Arabidopsis seed germination.

LRRop-1, induced by DOF6 transcription factor, negatively regulates abiotic stress responses during Arabidopsis seed germination. The lrrop-1 mutant has reduced ABA signaling, which is part of the underlying stress-remediation mechanism. The large family of leucine-rich repeat (LRR) proteins plays a role in plant immune responses. Most LRR proteins have multiple functional domains, but a subfamily is known to possess only the LRR domain. The roles of these LRR-only proteins in Arabidopsis remain largely uncharacterized. In the present study, we have identified 44 LRR-only proteins in Arabidopsis and phylogenetically classified them into nine sub-groups. We characterized the function of LRRop-1, belonging to sub-group V. LRRop-1 encodes a predominantly ER-localized LRR domain-containing protein that is highly expressed in seeds and rosette leaves. Promoter motif analysis revealed an enrichment in binding sites for several GA-responsive and stress-responsive transcription factors. The lrrop-1 mutant seeds showed enhanced seed germination on medium containing abscisic acid (ABA), paclobutrazol and NaCl compared to the wild type (WT), demonstrating higher abiotic stress tolerance. Also, the lrrop-1 mutant seeds have lower levels of endogenous ABA, but higher levels of gibberellic acid (GA) and jasmonic acid-Ile (JA-Ile) compared to the WT. Furthermore, lrrop-1 mutant seeds imbibed with ABA exhibited reduced expression of ABA-responsive genes compared to similarly treated WT seeds, suggesting suppressed ABA signaling events in the mutant. Furthermore, chromatin immunoprecipitation (ChIP) data showed that DNA BINDING1 ZINC FINGER6 (DOF6), a negative regulator of seed germination, could directly bind to the LRRop-1 promoter and up-regulate its expression. Thus, our results show that LRRop-1 regulates ABA-mediated abiotic stress responses during Arabidopsis seed germination.

Genomic Characterization of Endothelial Enhancers Reveals a Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene Expression.

Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

Histone Methyltransferase SETD2 Inhibits Tumor Growth <i>via</i> Suppressing CXCL1-Mediated Activation of Cell Cycle in Lung Adenocarcinoma

Background: The histone H3 lysine 36 methyltransferase SET-domain-containing 2 (SETD2) has been reported to be frequently mutated or deleted in many types of human cancer. However, the role of SETD2 in lung adenocarcinoma (LUAD) has not been well documented. Method: Real-time qPCR, western blotting analysis and immunohistochemistry (IHC) assays were utilized to analyze the expression pattern of SETD2 in LUAD tissues and cell lines. Cell viability analysis, colony formation and flow cytometry was used for cell growth analysis. RNA sequencing (RNA-seq) was used to analyze the downstream target gene of SETD2. Bioinformatics method was applied to analyze the regulatory network in which SETD2 and CXCL1 involved. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were utilized to test the interaction between SETD2 and CXCL1. Xenograft experiments were used to analyze tumor growth in vivo. Finding: In the present study, we found that SETD2 was significantly down-regulated both in LUAD tissues and cell lines. Functionally, the increased expression of SETD2 significantly attenuated the proliferation of cancer cells by affecting the cell cycle, whereas SETD2 deficiency dramatically improved these proliferative abilities of cancer cells. Through conjoint analysis of RNA-seq and ChIP data, we identified a functional target gene of SETD2, CXCL1, and its expression was negatively correlated with that of SETD2. Moreover, SETD2 deletion stimulated cell cycle-related proteins to promote LUAD. Further mechanistic studies demonstrated that histone H3 lysine 36 trimethylation (H3K36me3) catalyzed by SETD2 interacted with the promoter of CXCL1 to regulate its transcription and downstream signaling pathways, contributing to tumorigenesis in vitro and in vivo. Interpretation: Our findings suggested that SETD2 inhibited tumor growth via suppressing CXCL1-mediated activation of cell cycle, indicating that the regulation of H3K36me3 level by targeting SETD2 and/or the administration of downstream CXCL1 might represent a potential therapeutic way for new treatment in LUAD. Funding Statement: This study was supported by grants from the National Natural Science Foundation of China (31701111, 81902386), the National Key R&D Plan (2018YFC1313400), the National Key Technology R&D Program (2015BAI12B12), Postdoctoral Research Foundation of China (2018M642305), Natural Science Foundation of Jiangsu Province (BK20170295), Postdoctoral Science Foundation of Jiangsu Province (2018K029A) and Applied Basic Research Project of Changzhou (CJ20179024, CJ20190094). Declaration of Interests: The authors state that they have nothing to disclose. Ethics Approval Statement: All human tissue-related experiments were approved by the Ethical Committee of the Third Affiliated Hospital of Soochow University. Written informed consents were obtained from all patients before surgery. The animal-related experiments were approved by the Institutional Animal Care and Use Committee of the Third Affiliated Hospital of Soochow University.

HSF1Base: A Comprehensive Database of HSF1 (Heat Shock Factor 1) Target Genes.

HSF1 (heat shock factor 1) is an evolutionarily conserved master transcriptional regulator of the heat shock response (HSR) in eukaryotic cells. In response to high temperatures, HSF1 upregulates genes encoding molecular chaperones, also called heat shock proteins, which assist the refolding or degradation of damaged intracellular proteins. Accumulating evidence reveals however that HSF1 participates in several other physiological and pathological processes such as differentiation, immune response, and multidrug resistance, as well as in ageing, neurodegenerative demise, and cancer. To address how HSF1 controls these processes one should systematically analyze its target genes. Here we present a novel database called HSF1Base (hsf1base.org) that contains a nearly comprehensive list of HSF1 target genes identified so far. The list was obtained by manually curating publications on individual HSF1 targets and analyzing relevant high throughput transcriptomic and chromatin immunoprecipitation data derived from the literature and the Yeastract database. To support the biological relevance of HSF1 targets identified by high throughput methods, we performed an enrichment analysis of (potential) HSF1 targets across different tissues/cell types and organisms. We found that general HSF1 functions (targets are expressed in all tissues/cell types) are mostly related to cellular proteostasis. Furthermore, HSF1 targets that are conserved across various animal taxa operate mostly in cellular stress pathways (e.g., autophagy), chromatin remodeling, ribosome biogenesis, and ageing. Together, these data highlight diverse roles for HSF1, expanding far beyond the HSR.